A chip-based platform for the in vitro generation of tissues in three-dimensional organization

We describe a multi-purpose platform for the three-dimensional cultivation of tissues. The device is composed of polymer chips featuring a microstructured area of 1-2 cm(2). The chip is constructed either as a grid of micro-containers measuring 120-300 x 300 x 300 microm (h x l x w), or as an array of round recesses (300 microm diameter, 300 microm deep). The micro-containers may be separately equipped with addressable 3D-micro-electrodes, which allow for electrical stimulation of excitable cells and on-site measurements of electrochemically accessible parameters. The system is applicable for the cultivation of high cell densities of up to 8 x 10(6) cells and, because of the rectangular grid layout, allows the automated microscopical analysis of cultivated cells. More than 1000 micro-containers enable the parallel analysis of different parameters under superfusion/perfusion conditions. Using different polymer chips in combination with various types of bioreactors we demonstrated the principal suitability of the chip-based bioreactor for tissue culture applications. Primary and established cell lines have been successfully cultivated and analysed for functional properties. When cells were cultured in non-perfused chips, over time a considerable degree of apoptosis could be observed indicating the need for an active perfusion. The system presented here has also been applied for the differentiation analysis of pluripotent embryonic stem cells and may be suitable for the analysis of the stem cell niche.     

Determination of the microenvironment-pH and charge and size characteristics of amino acids through their electrophoretic mobilities determined by CZE

Effective electrophoretic mobility data of 20 amino acids reported in the literature are analyzed and interpreted through simple physicochemical models, which are able to provide estimates of coupled quantities like hydrodynamic shape factor, equivalent hydrodynamic radius (size), net charge, actual pK values of ionizing groups, partial charges of ionizing groups, hydration number, and pH near molecule (microenvironment-pH of the BGE). It is concluded that the modeling of the electrophoretic mobility of these analytes requires a careful consideration of hydrodynamic shape coupled to hydration. In the low range of pH studied here, distinctive hydrodynamic behaviors of amino acids are found. For instance, amino acids with basic polar and ionizing side chain remain with prolate shape for pH values varying from 1.99 to 3.2. It is evident that as the pH increases from low values, amino acids get higher hydrations as a consequence each analyte total charge also increases. This result is consistent with the monotonic increase of the hydrodynamic radius, which accounts for both the analyte and the quite immobilized water molecules defining the electrophoretic kinematical unit. It is also found that the actual or effective pK value of the alpha-carboxylic ionizing group of amino acids increases when the pH is changed from 1.99 to 3.2. Several limitations concerning the simple modeling of the electrophoretic mobility of amino acids are presented for further research.     

Distinct reaction pathways followed upon reduction of oxy-heme oxygenase and oxy-myoglobin as characterized by Mössbauer spectroscopy

Activation of O(2) by heme-containing monooxygenases generally commences with the common initial steps of reduction to the ferrous heme and binding of O(2) followed by a one-electron reduction of the O(2)-bound heme. Subsequent steps that generate reactive oxygen intermediates diverge and reflect the effects of protein control on the reaction pathway. In this study, M?ssbauer and EPR spectroscopies were used to characterize the electronic states and reaction pathways of reactive oxygen intermediates generated by 77 K radiolytic cryoreduction and subsequent annealing of oxy-heme oxygenase (HO) and oxy-myoglobin (Mb). The results confirm that one-electron reduction of (Fe(II)-O(2))HO is accompanied by protonation of the bound O(2) to generate a low-spin (Fe(III)-O(2)H(-))HO that undergoes self-hydroxylation to form the alpha-meso-hydroxyhemin-HO product. In contrast, one-electron reduction of (Fe(II)-O(2))Mb yields a low-spin (Fe(III)-O(2)(2-))Mb. Protonation of this intermediate generates (Fe(III)-O(2)H(-))Mb, which then decays to a ferryl complex, (Fe(IV)=O(2-))Mb, that exhibits magnetic properties characteristic of the compound II species generated in the reactions of peroxide with heme peroxidases and with Mb. Generation of reactive high-valent states with ferryl species via hydroperoxo intermediates is believed to be the key oxygen-activation steps involved in the catalytic cycles of P450-type monooxygenases. The M?ssbauer data presented here provide direct spectroscopic evidence supporting the idea that ferric-hydroperoxo hemes are indeed the precursors of the reactive ferryl intermediates. The fact that a ferryl intermediate does not accumulate in HO underscores the determining role played by protein structure in controlling the reactivity of reaction intermediates.     

Intramolecular carbozincation of unactivated alkenes occurs through a zinc radical transfer mechanism

The cyclizations of a number of terminally unsaturated alkenyl zinc iodides to cyclopentylmethylzinc iodides, formerly believed to be nonradical in nature, have been revealed as radical chain cyclizations initiated by adventitious oxygen. Five cases are presented in which the published carbozincation cis/trans selectivities are essentially the same as those found for the cyclizations of the unsaturated alkyl iodide precursors of the alkylzinc iodides by the iodine atom transfer method at approximately the same temperatures. In addition, it has been found that one of the organozinc cyclizations does not occur in a system in which oxygen has been rigorously excluded. The combined findings strongly suggest that these organozinc cyclizations occur by a zinc radical transfer mechanism rather than by a conventional carbometallation that is thought to occur with the analogous organolithium and organomagnesium cyclizations.     

Determination of chondroitin sulfate content in raw materials and dietary supplements by high-performance liquid chromatography with ultraviolet detection after enzymatic hydrolysis: single-laboratory validation

A method to quantify chondroitin sulfate in raw materials and dietary supplements at a range of about 5 to 100% (w/w) chondroitin sulfate has been developed and validated. The chondroitin sulfate is first selectively hydrolyzed by chondroitinase ACII enzyme to form un-, mono-, di-, and trisulfated unsaturated disaccharides; the resulting disaccharides are then quantified by ion-pairing liquid chromatography with ultraviolet detection. The amounts of the individual disaccharides are summed to yield the total amount of chondroitin sulfate in the material. Single-laboratory validation has been performed to determine the repeatability, accuracy, selectivity, limit of detection, limit of quantification, ruggedness, and linearity of the method. Repeatability precision for total chondroitin sulfate content was between 1.60 and 4.72% relative standard deviation, with HorRat values between 0.79 and 2.25. Chondroitin sulfate recovery from raw material negative control was between 101 and 102%, and recovery from finished product negative control was between 105 and 106%.